Research is in progress to investigate the conformation of eukaryotic messenger RNA using biophysical, chemical and enzymatic probes of structure. Data is obtained that suggest that specific, discrete-length, helical regions exist in rabbit and human globin mRNAs. Thermal melting studies demonstrate that the presence of Mg ions increases the stability of rabbit globin mRNA. The fraction of nucleotides in helical regions, measured by temperature induced hyperchromicity shifts, is sensitive to pH, with 55-60% and 70-75% of the bases in helical regions at pH 7.5 and 5.0, respectively. Carbodiimide reactions with non base-paired guanines and uracils in rabbit globin mRNA at different temperatures and in the presence of ethanol imply that stable secondary structure and weak tertiary interactions may co-exist in the molecule. Digestion of (125I)-labeled rabbit and human globin mRNAs with the single-strand specific nuclease S1 at pH 4.5 generates discrete RNA fragments between B-50 nucleotides in length, as observed on 20% acrylamide. 7M urea denaturing gels calibrated with markers of known length. Several S1 resistant fragments obtained by digestion of human sickle beta-globin mRNA are being sequenced to locate their position within the known primary sequence of this molecule.